<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> mechanosensitive channels: towards unpuzzling “glutamate efflux” for amino acid production

Corynebacterium glutamicum has been utilized for industrial amino acid production, especially for monosodium glutamate (MSG), the food-additive for the “UMAMI” category of taste sensation, which is one of the five human basic tastes. Glutamate export from these cells is facilitated by the opening of mechanosensitive channels in the cell membrane within the bacterial cell envelope following specific treatments, such as biotin limitation, addition of Tween 40 or penicillin. A long-unsolved puzzle still remains how and why C. glutamicum mechanosensitive channels are activated by these treatments to export glutamate. Unlike mechanosensitive channels in other bacteria, these channels are not simply osmotic safety valves that prevent these bacteria from bursting upon a hypo-osmotic shock. They also function as metabolic valves to continuously release glutamate as components of a pump-and-leak mechanism regulating the cellular turgor pressure. Recent studies have demonstrated that the opening of the mechanosensitive channel, MscCG, mainly facilitates the efflux of glutamate and not of other amino acids and that the “force-from-lipids” gating mechanism of channels also applies to the MscCG channel. The bacterial types of mechanosensitive channels are found in cell-walled organisms from bacteria to land plants, where their physiological functions have been specialized beyond their basic function in bacterial osmoregulation. In the case of the C. glutamicum MscCG channels, they have evolved to function as specialized glutamate exporters.     

Interactions of HP1 Bound to H3K9me3 Dinucleosome by Molecular Simulations and Biochemical Assays

Heterochromatin protein 1 (HP1), associated with heterochromatin formation, recognizes an epigenetically repressive marker, trimethylated lysine 9 in histone H3 (H3K9me3), and generally contributes to long-term silencing. How HP1 induces heterochromatin is not fully understood. Recent experiments suggested that not one, but two nucleosomes provide a platform for this recognition. Integrating previous and new biochemical assays with computational modeling, we provide near-atomic structural models for HP1 binding to the dinucleosomes. We found that the dimeric HP1α tends to bind two H3K9me3s that are in adjacent nucleosomes, thus bridging two nucleosomes. We identified, to our knowledge, a novel DNA binding motif in the hinge region that is specific to HP1α and is essential for recognizing the H3K9me3 sites of two nucleosomes. An HP1 isoform, HP1γ, does not easily bridge two nucleosomes in extended conformations because of the absence of the above binding motif and its shorter hinge region. We propose a molecular mechanism for chromatin structural changes caused by HP1.     

Molecular marker to identify the fungus gnat, <Emphasis Type="Italic">Bradysia</Emphasis> sp. (Diptera: Sciaridae), a new pest of Welsh onion and carrot in Japan

A new pest of Welsh onion and carrot, the dark-winged fungus gnat, Bradysia sp., was found in the northern region of Saitama Prefecture, Japan. DNA sequences of mitochondrial cytochrome c oxidase subunit I revealed that Bradysia sp. is identical to the Chinese chive gnat, Bradysia odoriphaga Yang and Zhang. This suggests that Bradysia sp. may be an invasive species that originates from continental East Asia. Direct sequencing analysis and PCR using a species-specific primer were found to be useful tools for discriminating Bradysia sp. from other native Japanese fungus gnats, such as Bradysia impatiens (Johannsen), Pnyxia scabiei (Hopkins), and Lycoriella ingenua (Dufour).     

High affinity sugar ligands of C-type lectin receptor langerin

Background

Langerin, a C-type lectin receptor (CLR) expressed in a subset of dendritic cells (DCs), binds to glycan ligands for pathogen capture and clearance. Previous studies revealed that langerin has an unusual binding affinity toward 6-sulfated galactose (Gal), a structure primarily found in keratan sulfate (KS). However, details and biological outcomes of this interaction have not been characterized. Based on a recent discovery that the disaccharide L4, a KS component that contains 6-sulfo-Gal, exhibits anti-inflammatory activity in mouse lung, we hypothesized that L4-related compounds are useful tools for characterizing the langerin-ligand interactions and their therapeutic application.

Characterization of three strains of Capnocytophaga canis isolated from patients with sepsis

Capnocytophaga canimorsus and Capnocytophaga cynodegmi, both commensal bacteria in the oral cavities of dogs and cats, are zoonotic pathogens. In particular, C. canimorsus causes sepsis and fatal septic shock. Recently, a novel Capnocytophaga species, C. canis, was isolated from the oral cavities of healthy dogs. It is reportedly oxidase‐negative and therefore considered avirulent in humans. In the present study, three strains of C. canis were isolated from Japanese patients with sepsis. All three strains, HP20001, HP33001 and HP40001, were oxidase‐positive. Nucleotide sequence identities of the 16S rRNA gene of the three strains to the C. canimorsus type strain ATCC35979, C. cynodegmi type strain ATCC49044 and C. canis type strain LMG29146 were 96.9–97.0%, 96.9–97.0% and 99.7–99.8%, respectively. Multi‐locus sequence analysis based on seven house‐keeping genes, dnaJ, fumC, glyA, gyrB, murG, trpB and tuf, revealed that the oxidase‐positive C. canis strains isolated in Japan and oxidase‐negative strains of C. canis from canine oral cavities in Switzerland were clustered in different genetic subgroups. These results indicate that the virulence of C. canis strains in humans is associated with oxidase activity.

     

Genetic characterization of hemagglutinin protein of measles viruses in Hokkaido district,Japan, 2006–2015

Strains of measles virus of genotypes D5, H1, D4, D8, and B3 were detected among epidemic, endemic, imported and import–associated cases in Hokkaido district, Japan, during 2006–2015. In the present study, their antigenic features were evaluated by determining the complete nucleotide sequences of their hemagglutinin proteins, which are a major target for neutralizing antibodies, and their amino acid sequences deduced. It was found that the hemagglutinin proteins of these strains had several novel amino acid changes in some functional regions. Although these strains have not caused further infections thus far, these antigenic changes should continue to be monitored to maintain their elimination status.

     

In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned,specific pathogen‐free Landrace piglets

Toll‐like receptor 5 is a pattern‐recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol‐killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen‐free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L‐3569 strain) to determine the effect of this specific SNP on ST infection in vivo. Eighteen ST‐infected piglets (six each with CC, CT, or TT) exhibited fever and diarrhea for 1 week after infection. TT piglets had the longest duration of fever. TT piglets had the greatest mean diarrhea score during the experimental period, followed by CT and CC piglets. Fecal ST shedding was greater in CT and TT pigs than CC pigs from 2 days after infection. Serum haptoglobin concentration increased in ST‐infected piglets and to greater extents in CT and TT pigs than CC pigs. Daily weight gain was lower in infected pigs, particularly TT piglets, than control pigs. To the best of our knowledge, this study is the first to demonstrate that impairment of TLR recognition affects pig susceptibility to disease in vivo. Thus, piglets with the T allele of swine TLR5 (C1205T) exhibit impaired resistance to ST infection. Furthermore, elimination of the T allele of this SNP from Landrace pigs would lead to enhancement of their resistance to ST infection.

     

Identification of proteins that bind to the neuroprotective agent neoechinulin A

Neoechinulin A is an indole alkaloid with several biological activities. We previously reported that this compound protects neuronal PC12 cells from cytotoxicity induced by the peroxynitrite generator 3-morpholinosydnonimine (SIN-1), but the target proteins and precise mechanism of action of neoechinulin A were unclear. Here, we employed a phage display screen to identify proteins that bind directly with neoechinulin A. Our findings identified two proteins, chromogranin B and glutaredoxin 3, as candidate target binding partners for the alkaloid. QCM analyses revealed that neoechinulin A displays high affinity for both chromogranin B and glutaredoxin 3. RNA interference-mediated depletion of chromogranin B decreased the sensitivity of PC12 cells against SIN-1. Our results suggested chromogranin B is a plausible target of neoechinulin A.     

Discovery of a novel 2-(1H-pyrazolo[3,4-b]pyridin-1-yl)thiazole derivative as an EP1 receptor antagonist and in vivo studies in a bone fracture model

We describe a medicinal chemistry approach to the discovery of a novel EP₁ antagonist exhibiting high potency and good pharmacokinetics. Our starting point is 1, an EP₁ receptor antagonist that exhibits pharmacological efficacy in cystometry models following intravenous administration. Despite its good potency in vitro, the high lipophilicity of 1 is a concern in long-term in vivo studies. Further medicinal chemistry efforts identified 4 as an improved lead compound with good in vitro ADME profile applicable to long term in vivo studies. A rat fracture study was conducted with 4 for 4?weeks to validate its utility in bone fracture healing. The results suggest that this EP₁ receptor antagonist stimulates callus formation and thus 4 has potential for enhancing fracture healing.